Method for producing citric acid

ABSTRACT

Method for producing citric acid which involves inoculating a citric acid-accumulating and hydrocarbon-assimilating strain of bacterium belonging to the genus Corynebacterium in an aqueous culture medium containing at least one C9-C20- normal paraffin as the principal carbon source; incubating the culture at a pH of from about 5 to about 8 until citric acid is substantially accumulated in the culture broth; and recovering the the soaccumulated citric acid therefrom. The advantage of the above method resides in the fact that the n-paraffin hydrocarbon source is available in large quantities and at low cost; the fermentation period (2-3 days) is considerably shorter than conventional methods and the citric acid thus-obtained is produced in a high yield and in a pure form.

United States Patent Fukuda et al.

[ 51 June 20, 1972 [54] METHOD FOR PRODUCING CITRIC ACID [73] Assignee: Takeda Chemical Industries, Ltd., Osaka,

Japan 22 Filed: Jan. 30, 1970 21 Appl.No.: 7,232

[51] Int. Cl. [58] Field of Search ..Cl2d 13/00 195/28, 42, 47, 3 H, 37

[56] References Cited OTHER PUBLICATIONS Kyowa Fermentation Ind. (1); Chemical Abs., 86,141K, page 8288, May 10, 1968 Tanaka, K.; Chem Abs, vol. 69, if 95103X, 1968 Kyowa Fermentation Ind. (11); Chem Abs., vol. 67, 72,4641(, 1967 Ladd, Aust. J of Biol. Sci., vol. 9, p. 92- 104, 1956 Primary ExaminerA. Louis Monacell Assistant Examiner-Gary M. Nath Artomeywenderoth, Lind & Ponack [57] ABSIRACT Method for producing citric acid which involves inoculating a citric acid-accumulating and hydrocarbon-assimilating strain of bacterium belonging to the genus Corynebacterium in an aqueous culture medium containing at least one C C, normal paraffin as the principal carbon source; incubating the culture at a pH of from about 5 to about 8 until citric acid is substantially accumulated in the culture broth; and recovering the the so-accumulated citric acid therefrom. The advantage of the above method resides in the fact that the n-paraifin hydrocarbon source is available in large quantities and at low cost; the fermentation period (2-3 days) is considerably shorter than conventional methods and the citric acid thus-obtained is produced in a high yield and in a pure form.

11 Claims, No Drawings containing, as the main carbon source, at least one normal paraffin containing nine to 20 carbon atoms in the molecule,"

incubating the culture at a pH of from about to about 8 until" citric acid is substantially accumulated in the culture broth;

and recovering so-accumulated citric acid therefrom. 1 Citric acid is in a great demand and used, for example, as am, acidulant in beverage and in pharmaceutical syrups. Regarding the production of citric acid by fermentation,; processes involving the use of microorganisms such as the molds belonging to the genera Penicillium, Aspergillus, etc. arei well known and have been the subjects of numerous reports; and patents. These processes, however, invariably depend; upon the employment of sugars and other expensive carbon; sources, and the fermentation period is as long as 5 to 12 days.? Recently, processes employing yeasts and bacteria have been disclosed, but the yields of citric acid' relative to the carbon sources used has never been as satisfactory as might have been expected. The present inventors have conducted an extensive study of fermentation with hydrocarbons as carbon sources and ultimately discovered that a group of bacteria, particularly those belonging to the genus Corynebacterium, produce citric acid from n-paraffins under aerobic conditions in unusually high yields. The present invention is the culmination of the abovefindings. From industrial and economic points of view, the method of this invention is by far superior to the conventional methods in that l hydrocarbons of n-paraflin series to be used as carbon sources are available in quantities and at low costs, (2) the fermentation period required is as short as 2 to 3 days, or less than a half of the time required in the prior ail, (3) the smaller amounts of impurities in the culture assist in the purification step, and (4) citric acid is accumulated in yields not less than the equivalent by weight of n-paraffin used as the carbon The main object of the present invention is to provide a method for producing citric acid in a good yield.

The other object of the present invention is to shorten the =1 fermentation period. The further object of the present invention is to facilitate the purification procedure and thus to provide an industrially i feasible method for producing citric acid. 5 These objects are realized by inoculating a citric acid-accui mulating and hydrocarbon-assimilating bacterium of the i genus Corynebacterium in an aqueous culture medium coni taining, as the main carbon source, at least one normal parafi fin containing nine to 20 carbon atoms in the molecule; incu- I ibating the culture at a pH of from about 5 to about 8 until icitric acid is substantially accumulated in the culture broth; and recovering so-accumulated citric acid therefrom. l The aforesaid bacterium of the genus Corynebacterium: iwhich is to be employed according to this invention, may any strain so long as it is capable of utilizing the hydrocarbon; mentioned above and converting the same to citric acid. The? following are a few typical examples which can be employed, 3 advantagi i har iiww ee, 1."

Corynebacterium sp. No. 1304 (ATCC 21461) Corynebacterium sp. No. 177 (ATCC 21458) Corynebacterium sp. No. 416 (ATCC 21459) Corynebacterium sp. No. 803 (ATCC 21460) Corynebacterium sp. No. 981 (ATCC 21462) Corynebacterium sp. No. 218 (ATCC 21463) Corynebacterium sp. No. l 17 (ATCC 21464) Corynebacterium sp. No. 279 (ATCC 21465) Corynebacterium sp. No. 384 (ATCC 21466) Corynebacterium sp. No. 628 (ATCC 21467) U.S.A., as may be witnessed by the listed ATCC members. The ATCC members of the bacterium contemplated in the present invention also appear inthe various Examples, etc., throughout the disclosure.

The microbiological characteristics ofthoee typical bacteria s 0 ur ;e. m w are shown in Iable l and Table 2. w TABLE 1 i Corynebacterium, sp. N o. 177 Corynebacterium, sp. No.416 Corynebacterium, sp. No. 981 Corynebacterium, sp. No. 218

ATCC 21458 ATCC 21459 A'ICO 21462 ATCC 21463 Shape andsize Rod-shaped, varying in size; Rod-shaped, varying in size; Rod-shaped, varying in size; Bod-shaped, varying in size;

comparatively long cells comparatively long cells comparatively long cells comparatively long cells are club-shaped. 0.7-0.9x are clubshaped. 0.8-0.9X are club-shaped. are club-shaped, some 1.2-1.8 1.3-1.8 curved. Non-motile Non-motile Non-motile Non-motile.

Asporogenous. Asporogenous Asporogenous Asporogenous. Positive Positive Positive Positive.

Medium growth; irregular teristies-bouillon margin and central conagar plate. vexity; opaque, dull; gray r with a yellowish cast. Bouillon agar slant Medium growth; beaded or echinulate; opaque, dull; gray with a yellowish cast, dry. Thick pellicle formed, transgray with a Medium growth; irregular margin; flat, opaque, dull; gray with a yellowish cat.

Medium growth; beaded or echinulate; opaque, dull;

yellowish cast, dry.

Good growth; irregular margin and convex; dry, opaque, dull; gray with a yellowish cast.

Good growth; beaded or echinulate; gray with a yellowish cast. yellowish cast.

Bouillon liquid Thick pellicle formed, trans- Thick pellicle formed, trans- Thick pellicle termed, transeulture. parent, with slight parent, with sediment. parent, with sediment. parent, with sediment.

sediment. Optimum tempera- 37 C 32 C 28 37 C 28-37 C.

was...

Corynebacterium, sp. No. 117 Corynebacterium, sp. N o. 279 Corynebacterium, sp. No. 384 Corynebacterium, sp. N o. 628

ATCC 21466 ATCC 21466 A'ICC 21467 ATCC 21464 Shape and size Rod-shaped, varying in size; Rod-shaped, varying in size; Rod-shaped, varying in size; Rod-shaped, varying in size;

comparatively long cells comparatively long cells comparatively long cells comparatively long cells are club-shaped. are cltb-shaped, some aire 0.80.9 (1.8-2.3u in are club-shaped. curve s ze.

Motility .4 Non-motile... Non-motile Non-motile Non-motile.

Sporulatiom. Asporogenous. Asporogenous. Asporogenous. Asporogenous.

Grams stain Positive Positive Positive Positive.

Cultural character- Good growth; irregular mar- Good growth; irregular mar- Good growth; irregular mar- Good growth; irregular rnaristies-bouillon gin and convex; dry, gin and hat; dry, opaque, gin and flat; dry, opaque, in and flat; dry, opaque, agar plate. opaque, dull; gray with a dull; gray with a yellowish dull; gray with a yellowish ull; gray with a yellowish yellowish cast. cast. cast. east.

Bouillon ngnr slnnt. Good growth; beaded or Good growth; echinulate; Good growth; echinulate; Good growth; beaded or echinulated; yellow-brown. gray with a yellowish cast. gray with a yellowish cast. eclfiinulafie, gray with a ye ow 5 cast. I

Bouillon liquid Thick pellicle formed, trans- Thick pellicle formed, trans- Thick pellicle formed, trans- Thick pellicle formed, transculture. parent, with sediment. parent, with sediment. parent, with sediment. parent, with sediment. =Optigmm 267 F 2837 C 37 C 28-37" C.

TABLE 1 (2) ('unlinllctl temperature. Optimum pH 6-8 6-8 6-8 TABLE 1 (3) Corynebacterium, sp. Coryncbacterium, sp. N0. 803 ATCC 21460 No. 1304 ATCC 21461 Shape and size.. Rod-shaped, varying in Rod-shaped, varying in size. Longer cells are size. Longer cells are clgb-shaped. 0.!)ll.1X cltb-shaped. 0.911.1X 2- t. t. Motility Nonotilo Non-motile. Sporulation- Asporogenous. Asporogenous. Gram's stain. Positive Positive. Bouillon agar Good growth; irregular Good growth; irregular plate. margin, dry, opaque, margin; umbilicatc, dry,

dul, light yellowish opaque, dull, gray with brown. a hint of yellow. Bouillon agar Good growth; beaded or Good growth; beaded or slant. echinulate, opaque, echinulate, opaque,

dull, light yellowish dull, gray with a hint brown. of y low. Bouillon liquid Thick pellicle formed; Thick pellicle formed;

culture. transparent, with slight transparent with slight sediment. sediment. Optimum 2832 O 2 C.

temperature. Optimum pH. 6-7 -9.

TABLE 2 Corynebacterium, Corynebacterium, sp. No. 177 Corynebacteriuni. sp. No. 416 Corynebacteriurn, sp. N 0. 803 sp. N o. 1304 Oxygen requirement Aerobic Aerobic Aerobic Aerobic. Litmus m Unchanged. Unchanged Alkalize Alkalized.

el Not liquefied Not liquefied. Not liquefied Not liquefied Produced. Produced Produced. iodueed Not formed-. Not formed Not formed... Not formed.

Not hydrolyz Not hydrolyzed Not hydrolyzed- Not hydrolyzed. Nitrates. Not reduced. Not reduced. Reduced Reduced. Catalasc test. Positive..-. Positive... Positive... Positive. Urease test. Positive Negative Positive Positive. sugar metabolism Acid produced anacio lea y Acid produced anaerobically" Unchanged Acid produced anaerobi- (Leiison method).

Fermentation of sugars- Acid production without visible gas formed.

Those bacteria are invariably club-shaped rods asporogenous, non-notile, gram-positive, aerobic and irregular in siie, and reference to Bergeys Manual of Dcterminative Bacteriol gy, 7th edition, suggests that they belong to the family of Corynebacteriacea.

The family of Corynebactcriacea includes the genera Corynebactcrium, Lysteria, Erysipelothrix, Microbacterium, Cellulomonas and Arthrobacter. However, bacteria of the genus Lysteria are motile, which bacteria of the genus Erysipelothrix make a feature of elongated filamentous cells and are weakly aerobic. Whereas bacteria of the genus Microbactcrium produce lactic acid and acidulate litmus milk, organisms of the genus Cellulomonas are capable of decomposing cellulose;

Thus, any of the above bacteria difiers from the bacteria used in this invention.

The foregoing result suggests that the bacteria according to this invention belong either to the genus Arthobacter or to the genus Corynebacterium.

There are differences between the two genera, however. In the first place, while young cells of bacteria of the genus Arthrobacter are gram-negative, their matured cells are grampositive. In other words they are gram-variants. Secondly, bacteria belonging to the genus Arthrobacter form characteristic spheroidal cells, in contrast to the bacteria according to this invention which are devoid of such a characteristic.

It is thus apparent that the present bacteria belong to the genus Corynebacterium.

Microorganisms, of course, are liable to undergo mutation, whether spontaneously or induced, and the present bacteria are no exception to the rule. It should be understood, however, that such variants and mutants may of course be employed in the method of this invention insofar as they are still capable of utilizing hydrocarbons to produce citric acid.

The culture medium to be employed in the present invention may vary with the strains employed, but such carbon sources as n-parafiins or various crude materials containing n parafi'ms. e.g. gas oil and heavy gas oil, can be employed Par- Acid production without visible gas formed.

Acid production without visible gas formed.

to make the concentration in the culture medium of the nor- I mal paraffin(s) with nine to 20 carbon atoms in the molecule, as a whole about 3 to 20 percent (volume/volume) in thc culture medium.

As these hydrocarbons are scarcely soluble in water, the addition thereof to an aqueous culture medium is practically carried out under stirring or shaking to prepare a suspension containing very fine particles. If desired, a suspending agent, c. g. a surfactant of the type of polyoxyethylcne sorbitan monostearate can be employed.

These hydrocarbons are by themselves sufiicient carbon sources, but, if desired, commonly cmployable carbon sources such as carbohydrate e.g. glucose) can be used together with the hydrocarbons.

As nitrogen sources, such inorganic ammonium salts as Nl-LCI, (NHJ SO (Nl-L),HPO,, Nl-LOl-l, Nl-LNO etc.,

urea, ammonium salts or organic acids, e.g. ammonium. acetate, and various organic nitrogenous materials such as dried yeast, yeast extract, meat extract, fish meal, soybean flour, corn steep liquor, peptone, distillers waste, etc. can be employed Those materials may be employed either singly or in combination.

if required, the inorganic salts which are conventionally employed, such as those of iron, manganese, calcium, magnesium, potassium, sodium, etc, as well as various nutrients may also be added to the medium.

The pH of the medium may be selected from thcwidc range which permits growth of the bacterium used. Generally the range of pH 5 to 8, particularly of pH 6 to 8, is preferred. If. in the course of cultivation, a drop in pH is induced by the production of citric acid, it is advisable to adjust the pH of the medium within the aforesaid range while continuing the cultivation. 1

For this purpose, the cultivation may be conducted with an occasional addition of a neutralizer which may, for example, be CaCO Ca(Ol-l) NH Ol-1, NaOH or Na- CO Altemative- 1y, to give an adequate bufi'ering action to the medium, CaCO for one, maybe preliminarily added in an amount commensurate with the possible pH drop.

While the incubation temperature may vary somewhat depending upon the particular bacterium used, the temperature is usually maintained within the range of 20 to 40 C.

1n carrying out the process of this invention, it is preferable to employ a liquid culture medium, and incubation is carried out aerobically, i.e. with aeration under static or submerged conditions.

In this operation, it is permissable to carry out defoaming as required, using any of such conventional defoaming agents as polyoxypropylene derivatives, soy bean oil, silicone oil, lard oil, etc.

According to this invention, the citric acid accumulated in the medium can be isolated and recovered by any per se conventional means.

Thus, such operations as neutralization, heating, cooling, precipitation, filtration, centrifugation, concentration, decolorization, crystallization and drying, as well as, if required, ion exchange resin treatments, can be carried out either singly or in combination. By such operations, the citric acid can be easily recovered in crystalline form.

The following examples are given to further illustrate this invention, but it is to be understood, however, that the scope of this invention is by no means limited thereto.

Throughout the specification, percentages are calculated on the weight per volume basis, and yields are calculated on weight of produced citric acid per weight of consumed normal paraffins.

The relationship between part(s) by weight and part(s) by volume is the same as hat between grarn(s) and milliliter(s).

EXAMPLE 1 A culture of Corynebacterium sp. No. 416 (ATCC 21459) is inoculated to 120 parts by volume of a medium (pl-1 7.0) which contains a petroleum fraction (4 percent) containing nparaffin (92 percent) having -12 carbon atoms, KH PO,( 0.2%), MgSO '7H' O (0.05%), FeSO,-7H O (0.02%), Nl-LCl (0.4%), yeast extract (0.1%) and CaCO; (3%) and incubated at 32 C. for 64 hours. The procedure yields 41.4mg. of citric acid per milliliter.

The culture is adjusted to pH 6.8 by the addition of 3N- NaOH. The broth is heated to 95 C. for about minutes, and then cooled to room temperature. The resulting sediment is recovered by filtration. The product is suspended in 50 parts by volume of water, followed by the addition of 5NH SO to adjust the suspension to pH 2.0. The sediment thus formed is filtered off, and the filtrate is concentrated under reduced pressure until a syrup of low consistency is obtained.

The syrup is allowed to stand in a cold room, whereuponcrystals of citric acid separate. The yield is 3.4 parts by weight s r ixd s- W- W EXAMPLE 2 A culture of Corynebacterium sp. No. 803 (ATCC 21460) is inoculated 40 parts by volume of a medium (pl-i 7.0) which contains a petroleum fraction (4%) containing n-parafiin (87%) having 16-20 carbon atoms, Kl-LPO, (0.2%), MgSO -7 H 0 (0.05%), MnSO,-7l-l O (0.002%), FeSO -7H O (0.01%), NH,NO;, (0.5%), urea (0.1%), dried yeast (0.05) and CaCo, (3%) and incubated at 28 C. for 3 days. The procedure yields 36mg. of citric acid PER milliliter of the broth. 100 parts by per of the broth is treated in the same manner as in Example 1, whereupon 3.24 pans by weight of citric acid is obtained by crystals.

EXAMPLE 3 A culture of Corynebacterium sp. No. 1304 (ATCC 21461) is inoculated to the same medium as one used in Example 2 and incubated at 32 C. for 2 days, whereupon 32mg. of citric acid is accumulated per milliliter of the broth. One hundred parts by volume of the broth is treated in the same manner as in Example 1.

The procedure yields 2.8 parts by weight of citric acid as crystals.

EXAMPLE 4 A culture of Corynebacterium sp. No. 803 (ATCC 21460) is inoculated to 120 parts by volume of a medium (pl-l 7.0) which contains n-octadecane (4%), Kl-1 PO, (0.2%), MgSO '7 H O (0.05%), MnSO,-7H O (0.001%), FeSO, (0.01%), Nl-LNO (0.5%), urea (0.1%) corn steep liquor (0.05%) and CaCO (3%) and incubated at 28 C. for 2 days. The procedure yields 44.2 mg of citric acid per milligram of the resulting broth.

One hundred parts by volume of the broth is treated in the same manner as in Example 1 to obtain 3.66 parts by weight of citric acid as crystals.

EXAMPLE 5 A culture of Corynebacterium sp. No. 177 (ATCC 21458) is inoculated in the same medium as that used in Example 1 and incubated at 34 C. for 3 days. The procedure yields a broth containing 24mg. of citric acid per milliliter. parts by volume of the broth is treated in the same manner as in Example 1 to obtain 1.7 part by weight of citric acid as crystals.

Example 6 Following cultures are inoculated to the same medium as used in Example 2 and incubated at 28 C. for 3 days, and the broth is treated in the same manner as in Example 1 Strain Accession 1. A method for producing citric acid which comprises inoculating a citric acid-accumulating and hydrocarbon-assimilating strain of Corynebacterium selected from the group consisting of sp. No. 177 (ATCC 21458), sp. No. 416 (ATCC 21459), sp. No. 803 (ATCC 21460), sp. No. 1304 (ATCC 21461 sp. No. 981 (ATCC 21462), sp. No. 21.8 (ATCC 21463), sp. No. 117 (ATCC 21464), sp. No. 279 (ATCC 21465), sp. No. 384 (ATCC 21466), sp. No. 628 (ATCC 21467) in an aqueous culture medium containing, as the main carbon source, at least one normal parafiin containing nine to 20 carbon atoms in the molecule; incubating the culture at a pH of from about 5 to about 8 until citric acid is substantially accumulated in the culture broth; and isolating the so-accumulated citric acid therefrom.

2. A method according to claim 1. wherein the bacterium is Corynebacterium sp. No. 177, ATCC 21458.

3. A method according to claim 1, wherein the bacterium is Corynebacterium sp. No. 416, ATCC 21459.

4. A method according to claim 1, wherein the bacterium is Corynebacterium sp. No. 1 l7, ATCC 2l464. Corynebacterium sp. No. 803, ATCC 21460. I 9. A method according to claim 1, wherein the bacterium is 5. A method according to claim 1, wherein the bacterium is Corynebacterium sp. No. 279, ATCC 21465. Corynebacterium sp. No. l304, ATCC 21461. 10. A method according to claim 1, wherein the bacterium 6. A method according to claim 1, wherein the bacterium is 5 is Corynebacterium sp. No. 384, ATCC 21466. Corynebacterium sp/ No. 981, ATCC 21462. 11. A method according to claim 1, wherein the bacterium 7. A method according to claim 1, wherein the bacterium is is Corynebacterium sp. No. 628, ATCC 21467. Corynebacterium sp. No. 218, ATCC 21463.

8. A method according to claim 1, wherein the bacterium is 

2. A method according to claim 1, wherein the bacterium is Corynebacterium sp. No. 177, ATCC
 21458. 3. A method according to claim 1, wherein the bacterium is Corynebacterium sp. No. 416, ATCC
 21459. 4. A method according to claim 1, wherein the bacterium is Corynebacterium sp. No. 803, ATCC
 21460. 5. A method according to claim 1, wherein the bacterium is Corynebacterium sp. No. 1304, ATCC
 21461. 6. A method according to claim 1, wherein the bacterium is Corynebacterium sp/ No. 981, ATCC
 21462. 7. A method according to claim 1, wherein the bacterium is Corynebacterium sp. No. 218, ATCC
 21463. 8. A method according to claim 1, wherein the bacterium is Corynebacterium sp. No. 117, ATCC
 21464. 9. A method according to claim 1, wherein the bacterium is Corynebacterium sp. No. 279, ATCC
 21465. 10. A method according to claim 1, wherein the bacterium is Corynebacterium sp. No. 384, ATCC
 21466. 11. A method according to claim 1, wherein the bacterium is Corynebacterium sp. No. 628, ATCC
 21467. 